I extracted RNA by GeneJET Plant RNA Purification Mini Kit from Thermoscientific and got 4 bands in undenatured electrophoresis (without DNAse treatment). Then I stored it in -20 °C. After about 24 hours, I ran that RNA sample again and got no band at all, only "smiley smear" (bottom picture). I don't know if my RNA got degraded or if there was any problem with electrophoresis.
Hello, for short-term storage (a few days for example) the RNA samples can be stored at –20°C if they are free of RNAse (that should be the case when using the GeneJET kit). So my guess would be a problem with the electrophoresis... I would recommend running a gel under denaturating conditions and check for the intensity of the rRNA bands.
Best regards
Hi, I agree with Christian. Short storage of RNA samples at -20 is fine. For me your RNA profile most likely is due to problems with electrophoresis.
I´m agree with Christian and Yordan, the "smiley smear" looks like to much RNA but another picture looks like was running in a gel for DNA, if your RNA is from leaf or complete plant you must see two bands corresponding to rRNA and litle bands of chloroplastics RNA, if not is fotosintetic tisue you must see only rRNA. Another thing to consider is the tissue from you extracted RNA, in been seeds is common later degradation. I´m stored RNA at –20°C for long-term without problem and I dont use any kit
Balint: I extracted RNA from rice leaves. Actually there are 4 bands in the above picture but they're not bright enough to be seen clearly and still a lot of genomic DNA. I still used that RNA samples for cDNA synthesis and the result was quites good. I wonder if precipiting RNA by ethanol for 30 minutes in -80 °C can improve the quality and quantity of RNA during extraction .
Upon freezing, the RNA and other solutes get concentrated and precipitated. Depending on RNA concentrations and a way how you melt the frozen solutions, the RNA and salts may not dissolve quickly and this process may continue after loading on the gel for analysis. Your second gel shows a distinctive high concentration fronts (smile-like patterns) usually observed when gels are overloaded. Try to dissolve your frozen RNA under higher temperature (e.g. 37oC) for longer time, vortex then spin before taking an aliquot for the gel analysis.
Balint: But I still got product from genomic DNA when I performed RT-PCR. The primers were designed to span an intron.The genomic DNA band appeared when I ran the RNA samples the second times (the bottom picture, very faint).I checked the RNA quality by Nanodrop, 260/280 values were about 2.04-2.1
The kit precipitated RNA with Ethanol 96%, I dont know if the result will be different if I use Ethanol 70%.
Contamination of the samples? Honestly, that seems quite unlikely to me. I would prefer Sergei's "gel overload / RNA not dissolved properly" hypothesis...
Hello Balint,
my explanation for the RNA smears would be: the RNA sample is pipetted into wells at one end of the gel. If these wells are filled with too much RNA, or if the sample is not properly diluted, the excess sample will not migrate according to its size but as a smear across the gel.
Best regards
Thanks for your explanation, Balint! Let us hope that Hoa is able to troubleshoot her problems with the RNA isolation/electrophoresis.
Best regards, Christian
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